N-ω-hydroxy-L-arginine derivatives for the treatment of diseases

ABSTRACT

The present invention relates to physically-chemically and pharmacokinetically enhanced N ω -hydroxy-L-arginine (NOHA) derivatives and a method for producing the NOHA derivatives having enhanced physical-chemical and pharmacokinetic properties according to the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application represents a National Stage application ofPCT/DE2010/000001 entitled “N^(ω)-Hydroxy-L-Arginine Derivatives for theTreatment of Diseases” filed Jan. 4, 2010, pending.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to physically-chemically andpharmacokinetically enhanced N^(ω)-hydroxy-L-arginine (NOHA) derivativesand a method for producing the NOHA derivatives having enhancedphysical-chemical and pharmacokinetic properties according to theinvention.

2. Discussion of the Prior Art

N^(ω)-hydroxy-L-arginine is the physiologically occurring intermediateof the NO synthase catalysed oxidation L-arginine. NOHA is oxidised in afurther step of NO synthase, nitrogen monoxide being released andL-citrulline being formed.

Thus the semi-essential amino acid L-arginine is the natural source forthe nitrogen monoxide (NO).

Nitrogen monoxide (NO) is of decisive importance among others forsupplying the organs with blood. NO leads indirectly to an increase ofthe vessels. The extent of the vascular dilation then has differenteffects in the individual organs. In the heart there is for example animproved circulation. In addition to expanding the vessels, NO also hasother properties:

-   -   The relaxing effect concerns not only the musculature of the        vessels but also that of the bronchial tree.    -   NO that is given off by the endothelial cells into the vessel        lumen can prevent the accumulation of blood platelets (=thrombus        formation).    -   In the nervous system, it is an important signalling substance        (transmitter) that influences the brain and gastrointestinal        functions. Thus nerve ends situated in the intestinal wall cause        a relaxation of the ring muscle due to the release of NO.    -   NO is formed in defence cells (macrophages) and is capable of        destroying bacteria. Stimulated by bacterial components (e.g.        lipopolysaccharides) macrophages produce NO in high        concentrations so that vital enzymes, e.g. those containing        iron, are blocked. The conservation of meat for example by        salting is based precisely on this process.

A dysregulation or reduced NO availability is thus related to diversecardiovascular diseases. A limited NO availability is thereforeassociated with the so-called endothelial dysfunction—a state ofmultifactorial genesis—that is associated with high blood pressure,atherosclerosis, arterial thrombosis, coronary heart disease, heartfailure, heart attack, hypercholesterolaemia and diabetes.

Old, rigid, atherosclerotically changed vessels can be deformed againwith NO. Blood circulation is thus improved and among others high bloodpressure can be normalised. For children that are born with seriousrespiratory dysfunction, the inhalation of NO is even today in usesuccessfully. NO promotes the erection of the penis which has led to thedevelopment of drugs against impotence (Viagra®).

Also for fighting tumors it is expected that new treatments can bepursued with NO since NO produced by white blood cells does not onlydestroy bacteria but also cells.

However, NO has quite a short life. Within a short period it reacts withoxygen molecules to form nitrite (NO₂ ⁻) and nitrate (NO₃ ⁻). The shortlife also explains why NO can only be formed directly at its target.

It is further known that NOHA is a potent inhibitor of arginase I with aK_(i) value between 30-42 μM.

In smaller in-vivo studies with rats, desired effects for treating theerectile dysfunction, endothelial dysfunction and hypertension couldalready be demonstrated using an i.v.-NOHA therapy.

Treating diseases associated with endothelial dysfunction, usingconventional NO donors has a few disadvantages. In the case of nitratesfor example the short therapeutic half time, the low oralbioavailability, partly adverse haemodynamic effects and toleranceeffects are to be mentioned in this context. Indeed, a proatherogeneeffect during long-term treatment using organic nitrates is beingdiscussed recently.

So that a therapy of NO-deficient diseases is possible with few sideeffects, the physiological situation has to be imitated as best aspossible, i.e. NO must only be released for a short time, in the rightamounts and of the right location. All these prerequisites are met usingNOHA as an NO-donor or, to take into consideration the time factor, witha retarding prodrug of NOHA. In addition to the fact that using thisstrategy, nitrogen monoxide is only released where it is needed and isformed to an extent that is too small, the fact can be exploited thatNOHA presents one of the most potent arginase inhibitors. Specificallyan increased arginase activity is discussed as a mechanism for reducedNO availability and thus as a contributing factor in the development ofthe endothelial dysfunction. Thus a dual mode of action is exploitedwhen using NOHA.

The use of L-arginine and N^(ω)-hydroxy-L-arginine and their simplecarboxylic acid esters, and also analogue N-hydroxyguanidine fortreating a multiplicity of diseases, have been described and patented (aselection: WO03045369, U.S. Pat. No. 6,277,884, WO0132167, CA02386938).

In practice, use of such compounds is limited by their badpharmacokinetic profile. The insufficient drug qualities of thissubstance can be explained above all by the presence of an unsubstitutedN-hydroxyguanidine function. In particular, the following effects can beobserved for the physical-chemical instability of N-hydroxyguanidines:

N-hydroxyguanidines decompose at room temperature and should be storedat 4-8° C., better −20° C. They are most stable in the form of theirsalts of strong acids. The following decomposition processes are known:

-   -   (1) Hydrolysis sensitivity: In particular at higher pH (>pH 7)        conversion takes place to cyanamides and hydrolysis to ureas,        which is relevant when taking into account the physiological pH        of 7.4 in vivo.    -   (2) Oxidation sensitivity: The susceptibility to oxidation is        probably the biggest issue of this class of substances since        using many and very different oxidation agents it could be shown        that two- and three-electron oxidation is possible. Using singly        substituted aliphatic and aromatic hydroxyguanidines, oxidation        potentials of E_(ox1)=+0.51-0.62 V and E_(ox2)=+1.14-1.81 V        could be determined. The decomposition products can differ as a        function of the oxidation agent. Also metal cations as for        example iron(II)(III) or copper(II) favour such processes or        participate in them. These processes are physiologically        relevant since it is known that oxidative processes dominate in        vivo and quite a range of physiological substances (reactive        oxygen species, metal cations) favour these oxidation processes.

Metabolic instability of N-hydroxyguanidines:

-   -   N-hydroxyguanidines are effectively reduced in the body in an        enzymatic catalysed manner to form corresponding guanidines. The        enzyme systems responsible for this have already been partly        identified. Thus the mitochondrial N-reduction is dependent on        cytochrome b5, cytochrome b5 reductase and a molybdenum        cofactor-dependent enzyme (mARC). The microsomale N-reduction is        catalysed by cytochrome b5, cytochrome b5-reductase and a third        component unknown so far.    -   In addition it is known that hydroxyguanidines can be        O-glucuronidised within the framework of a metabolic phase II        biotransformation to be converted into a form that can be        excreted more easily.

This leads to a strong limitation of the biological half time as aresult of thermal, hydrolytic, oxidative and enzymatic processes. Also ahigh first-pass effect is to be expected, considering the metabolicinstability that was mentioned.

SUMMARY OF THE INVENTION

It is therefore the object of the invention to improve thephysical-chemical and metabolic stability and thus the pharmacokineticproperties of N^(ω)-hydroxy-L-arginine and their simple carboxylic acidesters as well as analogue N-hydroxyguanidines.

It was surprisingly found that by substituting the hydroxyguanidinefunction according to the invention thermal and oxidation-stablederivatives of the NOHA were produced for the first time. FIG. 1 shows aschematic overview of the inventive substitution of the NOHAderivatives.

The chemical formula of the inventive derivatives is shown below.

The inventive substitutions are marked by X. Both a substitution at theoxygen of the hydroxylic groups X₂ leads to surprisingly stablederivatives, as also a substitution at the nitrogen X₁ and also acombination of the two inventive substitutions. In a usual way, thecarboxylic acid function can be esterified in a conventional manner withan alkyl or aryl radical or be present as an acid (R₁). Y can correspondto hydrogen, alkoxycarbonyl or aryloxycarbonyl radical. n can be 2 or 3.The chirality centre m can be R or S configured.

-   -   X_(1,2)=

-   -   X=Teil eines Ringsystems=

-   -   [X=part of ring system=]    -   where    -   R₁ can correspond to hydrogen, an alkyl or aryl radical; X₁ and        X₂ can be identical or different and correspond to the        structures (i-vii) (the exception is (i), where X₁ and X₂ have        to be different; exempt is also N^(ω)-hydroxy-L-arginine itself        and its carboxylic acid esters), where R₂, R₃ and R₄ can        correspond to hydrogen, alkyl or aryl radicals or can be shaped        such that their structure corresponds to a        monosaccharide(derivative) and R₅₋₁₂ can correspond to hydrogen,        an alkyl or aryl radical; n corresponds to a number of methyl        (CH₂) groups of 2-3; the chirality centre m can be R or S        configured; Y can correspond to hydrogen, an alkoxycarbonyl or        aryloxycarbonyl radical; Z can correspond to an oxygen or a        nitrogen atom; X_(1,2) are shaped such that they become part of        ring systems having the structure (viii) or (ix), where R₁₃ and        R₁₄ can correspond to hydrogen, alkyl or aryl radicals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a clear representation of the inventive optimisation of thedrug properties of N¹⁰⁷-hydroxy-L-arginine (NOHA) as NO donor andarginase inhibitor for treating diseases.

FIG. 2 For synthesising the inventive derivatives of theN¹⁰⁷-hydroxy-L-arginine a concept was developed that enables theillustration of very differently substituted compounds as represented bythis figure. As an example, not limiting the generality of theteachings, the synthesis of NOHA derivatives of different types aredescribed in the exemplary embodiments.

DETAILED DESCRIPTION OF THE INVENTION

Material and Methods

Exemplary Embodiment 1: O-Alkylised NOHA Derivatives

N^(ω)-benzyloxycarbonyl-N^(α)-(t-butyloxycarbonyl)-L-thiocitrulline-t-butylester(1)

General regulation in the style of Linton et al. [J. Org. Chem. 2000,65, 1566]:

7.0 mmol of N^(α)-(t-butyloxycarbonyl)-L-ornithine-t-butylester aredissolved in 250 mL of dry dichloromethane. The solution is cooled to 0°C. and 14 mL of a 0.5 M solution (in dichloromethane) ofbenzyloxycarbonylisothiocyanate (7.0 mmol) are added in drops over 30minutes. The reaction mixture is stirred for two hours, the solutionbeing warmed to room temperature. Using a rotary evaporator, the mixtureis concentrated to approximately one third of the original volume invacuum and washed in each case with 25 mL of 1% HCl, water and saturatedNaCl solution. The organic phase is dried over Na₂SO₄ and removed usingthe rotary evaporator. The thiourea 1 is mostly already >96% pure (DC)and is purified further using column chromatography.

After column chromatography over silica gel (cyclohexane/ethylacetate,4:1), a light-yellow oil is obtained that becomes solid in therefrigerator.

Yield: 3.13 g (93%)

TLC: R_(f)=0.31 (cyclohexane/ethylacetate, 4:1; ninhydrine)

¹H-NMR (CDCl₃):

δ/ppm=1.44, 1.46 (s, 9H, 2×C(CH₃)₃), 1.64-1.75 (m, 4H, β,γ-CH₂) 3.66 (m,2H, N—CH₂), 4.20 (m, 1H, α-CH), 5.08 (m, 1H, NH), 5.10 (s, 2H, CH₂-Cbz),7.36 (m, 5H, ArH), 8.15 (br s, 1H, NH), 9.65 (br s, 1H, NH).

¹³C-NMR (CDCl₃):

δ/ppm=24.8 (γ-CH₂), 28.7, 29.0 (2×C(CH₃)₃), 30.9 (β-CH₂), 45.8 (N—CH₂),54.2 (α-CH), 68.8 (CH₂-Cbz), 80.4, 82.8 (2×C(CH₃)₃), 129.0, 129.4, 129.5(ArCH), 135.2 (ArC), 153.2 (CO-Cbz), 156.0 (CO-Boc), 172.2 (COO^(t)Bu),179.8 (C═S).

MS (ESI):

m/z=482 [M+H]⁺, 426 [M−C₄H₈+H]⁺, 370 [M−2×C₄H₈+H]⁺, 326[M−2×C₄H₈−CO₂+H]⁺.

C₂₃H₃₅N₃O₆S (481.61)

Calculated C 57.36 H 7.33 N 8.73 Found C 57.55 H 7.60 N 8.68

N^(α)-(t-butyloxycarbonyl)-N^(ω)-ethoxycarbonyl-L-thiocitrulline-t-butylester(2)

8.0 mmol of N^(α)-(t-butyloxycarbonyl)-L-ornithine-t-butylester aredissolved in 250 mL of dry dichloromethane. The solution is cooled to 0°C., and 927 mg ethoxycarbonylisothiocyanate (7.0 mmol), dissolved in 15mL of dry dichloromethane, are added drop-wise over a period of 30minutes. The reaction mixture is stirred at room temperature for twohours. The mixture is then concentrated using the rotary evaporator toapproximately one third of the original volume and washed with in eachcase 25 mL of 1% HCl, water and saturated NaCl solution. The organicphase is dried over Na₂SO₄ and concentrated in vacuum. The thiourea 2 atthis point is mostly already >than 96% pure (DC). The product is startedto be dissolved in the solvent, activated carbon is added to it and itis cleaned by means of flash chromatography over a short silica gelcolumn. A column with about 20 g of silica gel is used, and the eluentis cyclohexane/ethylacetate (4:1).

Yield: 2.76 g of a colourless oil (94%)

TLC: R_(f)=0.31 (cyclohexane/ethylacetate, 4:1; ninhydrine)

Melting point: 81° C.

¹H-NMR (CDCl₃):

δ/ppm=1.31 (t, 3H, ³J=7.1 Hz, CH₂—CH₃), 1.45, 1.47 (2×s, 9H, C(CH₃)₃,1.61-1.90 (m, 4H, β,γ-CH₂), 3.66 (pseudo q, 2H, N—CH₂), 4.22 (q, 2H,³J=7.1 Hz, CH₂—CH₃), 5.08 (m, 1H, α-CH), 8.06, 9.70 (2×br s, 1H, NH).

¹³C-NMR (CDCl₃):

δ/ppm=14.9 (CH₂—CH₃), 24.9 (γ-CH₂), 28.7, 29.0 (2×C(CH₃)₃), 31.0(β-CH₂), 45.8 (N—CH₂), 54.3 (α-CH), 63.4 (O—CH₂), 82.8 (C(CH₃)₃), 153.4(CO-Boc), 156.2 (CO-Boc), 172.5 (COO^(t)/Bu), 180.1 (C═S).

MS (ESI):

m/z=442 [M+Na]⁺, 420 [M+H]⁺, 308 [M−2×C₄H₈+H]⁺, 264 [M−2×C₄H₈—CO₂+H]⁺.

C₁₈H₃₃N₃O₆S (419.54)

Calculated C 51.53 H 7.93 N 10.02 S 7.64 Found C 51.68 H 7.97 N 10.06 S7.62

N^(ω)-benzyloxycarbonyl-N^(α)-(t-butyloxycarbonyl)-N^(ω)-methoxy-L-arginine-t-butylester(3)

0.5 mmol of the thiourea 1 are dissolved in 5 mL of dry dichloromethaneand 522 μL DIPEA (3 mmol) and 1.5 mmol of hydroxylammonia hydrochlorideis added. The solution is brought to 0° C. for about 30 minutes and 287mg N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.5mmol) are added. Unless specified, the mixture is stirred over night atroom temperature. The solution is diluted with approximately 10 mL ofdichloromethane and washed in, each case with 5 mL of 1% HCl, water andsaturated NaCl solution. The organic phase is dried over Na₂SO₄ andconcentrated using the rotary evaporator. The result is mostly oils thatare further purified using flash chromatography over, silica gel. Theeluents used and the yields that were achieved are specified with therespective substances.

The eluent used is dichloromethane/methanol (99:1).

Yield: 235 mg of a colourless oil (95%)

TLC: R_(f)=0.30 (dichloromethane/methanol, 99:1; ninhydrine)

¹H-NMR (CDCl₃):

δ/ppm=1.44, 1.46 (2×s, 9H, C(CH₃)₃), 1.58-2.02 (m, 4H, β,γ-CH₂), 3.11(m, 2H, N—CH₂), 3.66 (s, 3H, O—CH₃), 4.18 (m, 1H, α-CH), 5.11 (m, 1H,NH), 5.13 (s, 2H, CH₂-Cbz), 6.25 (m, 1H, NH), 7.36 (m, 5H, ArH), 7.91(br s, 1H, NH).

¹³C-NMR (CDCl₃):

δ/ppm=25.6 (γ-CH₂), 28.7, 29.0 (2×C(CH₃)₃), 30.9 (β-CH₂), 41.2 (N—CH₂),54.5 (α-CH), 62.0 (O—CH₃), 68.3 (CH₂-Cbz), 80.3, 82.5 (2×C(CH₃)₃),129.0, 129.3, 129.4 (ArCH), 135.8 (ArC), 148.8 (C═N), 153.6 (CO-Cbz),156.0 (CO-Boc), 172.5 (COO^(t)Bu).

MS (ESI):

m/z=517 [M+Na]⁺, 495 [M+H]⁺, 439 [M−C₄H₈+H]⁺.

C₂₄H₃₈N₄O₇ (494.58)

Calculated C 58.28 H 7.74 N 11.33 Found C 58.72 H 7.99 N 11.22

N^(ω)-methoxy-L-arginine Bis(trifluoracetate) (3)

The protected L-arginine 3 (0.4 mmol) is stirred in 8 mL of TFA and 2.4mL of thioanisol for 30 minutes at room temperature. Then the majorityof TFA is distilled off in vacuum, and 5 mL of water and 15 mL ofdiethylether are added. The organic phase is also extracted twice using5 mL of water, and the combined aqueous phases are finally washed with 5mL of diethylether. The aqueous phase is concentrated using the rotaryevaporator (at approximately 35° C.) and taken up with little 0.1% ofTFA (in Aqua bidest.). Flash chromatography follows over an RP-18 column(eluent: 0.1% of TFA_((aq))), ninhydrine-positive fractions beingcombined. The combined fractions are concentrated to a volume ofapproximately 10 mL using the rotary evaporator and then lyophilisedwith freeze drying.

Yield: 171 mg of a colourless oil (99%), R_(f)=0.56(i-propanol/water/acetic acid, 6:3:1; ninhydrine)

¹H-NMR (D₂O):

δ/ppm=1.78 (m, 2H, γ-CH₂), 1.99 (m, 2H, β-CH₂), 3.31 (t, 2H, ³J=6.8 Hz,N—CH₂), 3.75 (s, 3H, O—CH₃), 4.06 (t, ³J=6.2 Hz, 1H, α-CH).

¹³C-NMR (D₂O, TPS):

δ/ppm=26.4 (γ-CH₂), 29.7 (β-CH₂), 43.0 (N—CH₂), 55.5 (α-CH), 67.3(O—CH₃), 160.1 (C═N), 174.8 (CO).

HRMS (m/z):

calculated for C₇H₁₇N₄O₃ [M+H]⁺=205.12952, found: 205.12951.

N^(ω)-methoxy-L-arginine-ethylester dihydrochloride (4)

For the esterification, 238 mg of the free amino acid 3 (0.55 mmol) aredissolved in 5 mL of absolute ethanol in an argon atmosphere. Thesolution is stirred for 30 minutes at −10° C. before HCl gas isintroduced into the solution for approximately 5-10 minutes. The batchis then stirred further for an hour at 0° C. and placed in therefrigerator for 36 hours. The solution is carefully concentrated atroom temperature in vacuum and lyophilised. The product thus obtained isa very hygroscopic, amorph solid which liquefies on contact with air.

Yield: 168 mg of a clear oil (99%)

TLC: R_(f)=0.18 (i-propanol/water/acetic acid, 8:1:1; ninhydrine)

¹H-NMR (DMSO-d₆):

δ/ppm=1.24 (t, ³J=7.2 Hz, 3H, CH₂—CH), 1.47-1.90 (m, 4H, β,γ-CH₂), 3.22(m, 2H, N—CH₂), 3.64 (s, 3H, O—CH₃), 3.99 (m, 1H, α-CH), 4.21 (q, ³J=7.2Hz, 2H, CH₂—CH₃), 8.04 (br s, 2H, NH₂), 8.31 (br t, 1H, NH), 8.72 (br s,3H, NH₃ ⁺), 11.33 (br s, 1H, NH⁺).

¹³C-NMR (DMSO-d₆):

δ/ppm=13.9 (CH₂—CH₃), 24.0 (γ-CH₂), 27.0 (β-CH₂), 40.0 (N—CH₂), 51.4(α-CH), 61.7 (CH₂—CH₃), 64.4 (O—CH₃), 157.2 (C═N), 169.2 (CO).

HRMS (m/z):

calculated for C₉H₂₁N₄O₃ [M+H]⁺=233.16082, found: 233.16064.

C₉H₂₀N₄O₃.2.0HCl.0.7H₂O (317.82)

Calculated C 34.01 H 7.42 N 17.63 Found C 33.65 H 7.66 N 18.20

N^(α)-(t-butyloxycarbonyl)-N^(ω)-ethoxycarbonyl-N^(ω′)-methoxy-L-arginine-t-butylester(5)

210 mg of the thiourea 2 (0.5 mmol) are dissolved in 5 mL of drydichloromethane and 261 μL of DIPEA (1.5 mmol) and 62.6 mg ofmethoxylamine hydrochloride (0.75 mmol) are added. The solution isbrought to 0° C. for approximately 30 minutes and 143.5 mg of EDCl (0.75mmol) are added. The mixture is stirred at room temperature over night.The solution is diluted with approximately 10 mL of dichloromethane andwashed in each case with 5 mL of 1% HCl, water and saturated NaClsolution. The organic phase is dried over Na₂SO₄ and concentrated invacuum. The raw product is further purified by means of columnchromatography over silica gel (dichloromethane/methanol, 98:2).

Yield: 203 mg of a colourless oil (94%)

TLC: R_(f)=0.26 (dichloromethane/methanol, 98:2; ninhydrine)

¹H-NMR (CDCl₃):

δ/ppm=1.27 (t, ³J=7.1 Hz, 3H, CH₂—CH₃), 1.43, 1.45 (2×s, 9H, C(CH₃)₃),1.56-1.89 (m, 4H, β,γ-CH₂), 3.09 (m, 2H, N—CH₂), 3.66 (s, 3H, O—CH₃),4.16 (br q, ³J=7.1 Hz, 3H, CH₂—CH₃, α-CH), 5.10, 6.26 (2×br m, 1H, NH),7.80 (br s, 1H, NH).

¹³C-NMR (CDCl₃):

δ/ppm=14.9 (CH₂—CH₃), 25.6 (γ-CH₂), 29.7 (β-CH₂), 29.0, 30.9(2×C(CH₃)₃), 41.2 (N—CH₂), 54.5 (α-CH), 62.0 (CH₂—CH₃), 62.6 (O—CH₃),80.2, 82.5, (2×C(CH₃)₃), 149.0 (C═N), 153.8 (CO-Eoc), 156.0 (CO-Boc),172.4 (COO^(t)Bu).

MS (ESI ):

m/z=455 [M+Na]⁺, 433 [M+H]⁺, 377 [M−C₄H₈+H]⁺.

C₁₉H₃₆N₄O₇ (432.52)

Calculated C 52.76 H 8.39 N 12.95 Found C 53.65 H 8.57 N 13.24

N^(ω)-ethoxycarbonyl-N^(ω′)-methoxy-L-arginine Bis(trifluoracetate) (6)

200 mg of the completely protected A′ -methoxy-L-arginine 5 (0.46 mmol)are stirred in 5 mL of TFA at first for 30 minutes at 0° C. and then forthree hours at room temperature. TFA is carefully distilled off at thelowest possible temperature in vacuum, and the residue is taken up witha little Aqua bidest. There follows a further purification by means offlash chromatography on an RP-18 column (0.1% of TFA in Aqua bidest.).The ninhydrine-positive fractions are combined, concentrated using therotary evaporator at approximately 30° C. to a residual volume ofapproximately 10 mL and lyophilised.

Yield: 225 mg of a colourless oil (97%)

TLC: R_(f)=0.44 (i-propanol/water/acetic acid, 8:1:1, ninhydrine)

¹H-NMR (DMSO-d₆):

δ/ppm=1.22 (t, ³J =7.1 Hz, 3H, CH₂—CH₃), 1.50-1.87 (m, 4H, β,γ-CH₂),3.12 (br t, 2H, N—CH₂), 3.62 (s, 3H, O—CH₃), 3.90 (m, 1H, α-CH), 4.13(q, ³J=7.1 Hz, 2H, CH₂—CH₃), 7.35 (br s, 1H, NH), 8.28 (br s, 3H, NH₃⁺).

¹³C-NMR (DMSO-d₆):

δ/ppm=14.1 (CH₂—CH₃), 24.6 (γ-CH₂), 27.3 (β-CH₂), 40.6 (N—CH₂), 51.7(α-CH), 61.6 (CH₂—CH₃), 62.1 (O—CH₃), 149.0 (C═N), 153.5 (CO-Eoc), 170.9(CO).

HRMS (m/z):

calculated for C₁₀H₂₁N₄O₅ [M+H]⁺=277.15065, found: 277.15049.

C₁₀H₂₀N₄O₅.2.0 CF₃COOH.0.4 H₂O (513.56)

Calculated C 32.74 H 4.87 N 10.91 Found C 32.44 H 4.69 N 10.53

N^(ω)-ethoxycarbonyl-N^(107 ′)-methoxy-L-arginine-ethylesterdihydrochloride (7)

For the esterification, 200 mg of the free amino acid 6 (0.397 mmol) aredissolved in 5 mL of absolute ethanol in an argon atmosphere. Thesolution is stirred at −10° C. for 30 minutes, before HCl gas isintroduced into the solution for approximately 5-10 minutes. The batchis then stirred further for an hour at 0° C. and placed in therefrigerator for 36 hours. The solution is carefully concentrated invacuum at room temperature and lyophilised. The product thus obtained isa very hygroscopic, amorph solid that liquefies on contact with air.

Yield: 150 mg of a colourless oil (99%)

¹H-NMR (DMSO-d₆):

δ/ppm=1.23, 1.25 (2×t, ³J=7.0 Hz, 3H, CH₂—CH), 1.55-1.88 (m, 4H,β,γ-CH₂), 3.32 (br t, 2H, N—CH₂), 3.71 (s, 3H, O—CH₃), 3.95 (m, 1H,α-CH), 4.19 (m, 4H, 2×CH₂—CH₃), 8.80 (br s, 4H, NH₃ ⁺, NH).

¹³C-NMR (DMSO-d₆):

δ/ppm=13.9, 14.0 (2×CH₂—CH₃), 24.0 (γ-CH₂), 26.9 (β-CH₂), 41.1 (N-CH₂),51.4 (α-CH₂), 61.7, 62.5 (2×CH₂—CH₃), 63.9 (O—CH₃), 150.7 (C═N), 152.6(CO-Eoc), 169.2 (COOEt).

HRMS (m/z):

calculated for C₁₂H₂₅N₄O₅ [M+H]⁺=305.18195, found: 305.18176.

C₁₂H₂₄N₄O₅.2.0 HCl.0.6 H₂O (388.08)

Calculated C 37.14 H 7.06 N 14.44 Found C 36.67 H 7.59 N 15.00Exemplary Embodiment 2: O-carboxyalkylated NOHA Derivatives

N^(ω)-benzyloxycarbonyl-N^(α)-(t-butyloxycarbonyl)-N^(ω′)-(methoxycarbonyl)methoxy-L-arginine-t-butylester(8)

241 mg of the thiourea 1 (0.5 mmol) are dissolved in 5 mL of drydichloromethane and 261 μL of DIPEA (0.75 mmol) and 79 mg ofaminooxyacetic acid methylester (0.75 mmol) are added. The solution isbrought to 0° C. for approximately 30 minutes and 143.5 mg of EDCl (0.75mmol) are added. The mixture is stirred over night at room temperature.The solution is diluted with approximately 10 mL of dichloromethane andwashed in each case with 5 mL of 1% HCl, water and saturated NaClsolution. The organic phase is dried over Na₂SO₄ and concentrated invacuum. The raw product is purified further by means of chromatographyover silica gel (cyclohexane/ethylacetate, 3:2).

Yield: 254 mg of a colourless oil (92%) TLC: R_(f)=0.48(cyclohexane/ethylacetate, 3:2; ninhydrine)

¹H-NMR (CDCl₃):

δ/ppm=1.44, 1.46 (2×s, 9H, C(CH₃)₃), 1.50-1.87 (m, 4H, β,γ-CH₂), 3.07(m, 2H, N—CH₂), 3.73 (s, 3H, O—CH₃), 4.16 (m, 1H, α-CH), 4.41 (s, 2H,O—CH₂), 5.08 (m, 1H, NH), 5.15 (s, 2H, CH₂-Cbz), 6.37 (br t, ³J=5.3 Hz,1H, NH), 7.30-7.39 (m, 5H, ArH), 8.23 (br s, 1H, NH).

¹³C-NMR (CDCl₃):

δ/ppm=25.5 (γ-CH₂), 28.7 (β-CH₂), 29.0, 30.9 (2×C(CH₃)₃), 41.2 (N—CH₂),52.4 (O—CH₃), 54.5 (α-CH), 68.3 (CH₂-Cbz), 71.2 (O—CH₂), 80.3, 82.5(2×C(CH₃)₃), 129.0, 129.28, 129.34 (ArCH), 135.9 (ArC), 150.8 (C═N),153.7 (CO-Cbz), 156.0 (CO-Boc), 171.7, 172.4 (COO^(t)Bu, COOMe).

MS (ESI):

m/z=575 [M+Na]⁺, 553 [M+H]⁺, 497 [M−C₄H₈+H]⁺.

N^(ω)-carboxymethoxy-L-arginine dihydrochloride (9)

270 mg of the completely protected N^(ω)-carboxymethoxy-L-arginine 8(0.489 mmol) are stirred at 50-60° C. for four hours in 5 mL of 6 N HCl.The batch is concentrated in vacuum, approximately 1-2 mL of Aquabidest. are added, and it is then purified by means of flashchromatography on an RP-18 column (0.1% of TFA in Aqua bidest.).Ninhydrine-positive fractions are combined, concentrated using therotary evaporator at 30° C. to only a few milliliters and thenlyophilised.

Yield: 150 mg of a white, amorphous solid (96%)

TLC: R_(f)=0.53 (i-propanol/water/acetic acid, 6:3:1; ninhydrine) ¹H-NMR(D₂O):

δ/ppm=1.76-2.18 (m, 4H, β,γ-CH₂), 3.41 (br t, ³J=6.7 Hz, 2H, N—CH₂),4.17 (br t, ³J=6.2 Hz, α-CH), 4.63 (s, 2H, O—CH₂).

¹³C-NMR (D₂O, TPS):

δ/ppm=26.3 (γ-CH₂), 29.6 (β-CH₂), 43.2 (N—CH₂), 55.3 (α-CH), 75.5(O—CH₂), 161.0 (C═N), 174.6, 175.3 (2×CO).

MS (ESI):

m/z=249 [M+H]⁺.

C₈H₁₆N₄O₅.2.0 HCl.0.5 H₂O (330.17)

Calculated C 29.10 H 5.80 N 16.97 Found C 29.00 H 5.99 N 17.16

N^(α)-(t-butyloxycarbonyl)-N^(ω)-ethoxycarbonyl-N^(ω′)-(ethoxycarbonyl)methoxy-L-arginine-t-butylester(10)

The preparation and working-up take place under the same conditions asdescribed for the analogous compound 8, with the thiourea 2 andaminooxyacetic acid ethylester as starting substances (batch size: 1.0mmol).

Yield: 500 mg of a colourless oil (99%)

TLC: R_(f)=0.51 (cyclohexane/ethylacetate, 3:2; ninhydrine)

¹H-NMR (CDCl₃):

δ/ppm=1.26 (t, ³J=7.1 Hz, 3H, CH₂—CH₃), 1.27 (t, ³J=7.1 Hz, 3H, CH₂-CH),1.42, 1.44 (2×s, 9H, C(CH₃)₃), 1.51-1.85 (m, 4H, β,γ-CH₂), 3.06 (m, 2H,N—CH₂), 4.16 (q, ³J=7.11 Hz, 2H, CH₂—CH₃), 4.19 (q, ³J=7.17 Hz, 2H,CH₂—CH₃), 4.39 (s, 2H, O—CH₂), 5.08 (m, 1H, NH), 6.40 (br t, 1H, NH),8.19 (br s, 1H, NH).

¹³C-NMR (CDCl₃):

δ/ppm=14.1, 14.2 (2×CH₂—CH₃), 24.8 (γ-CH₂), 27.9, 28.3 (233 C(CH₃)₃),30.2 (β-CH₂), 40.5 (N—CH₂), 53.7 (α-CH), 60.8, 61.9 (2×CH₂—CH₃), 70.7(O—CH₂), 79.5, 81.8 (2×C(CH₃)₃), 150.4 (C═N), 153.2 (CO-Eoc), 155.3(CO-Boc), 170.7,. 171.7 (COOEt, COO^(t)Bu).

MS (ESI):

m/z=527 [M+Na]⁺, 505 [M+H]⁺, 449 [M−C₄H₈]⁺.

C₂₂H₄₀N₄O₉ (504.59)

Calculated C 52.37 H 7.99 N 11.10 Found C 53.09 H 7.90 N 11.44

N^(ω)-ethoxycarbonyl-N^(ω′)-(ethoxycarbonyl)methoxy-L-argininebis(trifluoracetate) (11)

252 mg of the completely protected precursor 10 (0.5 mmol) are stirredin 5 mL of TFA at first at 0° C. for 30 minutes and then at roomtemperature for three hours. TFA is carefully distilled off at thelowest possible temperature in vacuum, and the residue is taken up witha little Aqua bidest. There follows the further purification by means offlash chromatography on an RP-18 column (0.1% TFA_((aq))/methanol stepgradient, 5-30%). The ninhydrine-positive fractions are combined,concentrated using the rotary evaporator at approximately 30° C. to aresidual volume of approximately 10 mL and lyophilised.

Yield: 277 mg of a clear oil (96%)

TLC: R_(f) =0.62 (i-propanol/water/acetic acid, 8:1:1; ninhydrine)

The substance is present as a solvent in DMSO-d₆ as an isomer mixture ina ratio of approximately 8.6:1.4 (at 300 K, relative to the singulet ofCH₂ of the ethoxycarbonylmethoxy radical). The shifts specified refer tothe main isomer.

¹H-NMR (DMSO-d₆):

δ/ppm=1.19 (t, ³J=7.10 Hz, 3H, CH₂—CH 1.21 (t, ³ J=7.08 Hz, 3H,CH₂—CH₃), 1.48-1.87 (m, 4H, β,γ-CH₂), 3.02 (m, 2H, N—CH₂), 3.89 (m, 1H,α-CH), 4.10 (q, ³J=7.04 Hz, 2H, CH₂—CH₃), 4.12 (q, ³J=7.13 Hz, 2H,CH₂—CH₃), 4.37 (s, 2H, O—CH₂), 6.54 (br s, 1H, NH), 8.24 (br s, 3H, NH₃⁺), 10.64 (br s, COOH).

¹³C-NMR (DMSO-d₆):

δ/ppm=14.0, 14.2 (2×CH₂—CH₃), 27.4 (CH₂), 40.3 (N—CH₂), 51.8 (α-CH),60.2, 61.3 (2×CH₂—CH₃), 70.3 (O—CH₂), 169.9, 171.0 (3×CO).

HRMS (m/z):

calculated for C₁₃H₂₅N₄O₇ [M+H]⁺=349.17178, found: 349.17155.

C₁₃H₂₄N₄O₇.3.2 CF₃COOH.1.5 H₂O (740.26)

Calculated C 31.48 H 4.11 N 7.57 Found C 31.52 H 4.24 N 7.40

N^(ω)-ethoxycarbonyl-N^(ω′)-(ethoxycarbonyl)methoxy-L-arginine-ethylesterdihydrochloride (12)

For the esterification, 288 mg of the free amino acid 11 (0.5 mmol) aredissolved in an argon atmosphere in 5 mL of absolute ethanol. Thesolution is stirred at 31 10° C. for 30 minutes, before HCl gas isintroduced into the solution for approximately 5-10 minutes. The batchis then stirred further for an hour at 0° C. and placed in therefrigerator over night. The solution is carefully concentrated invacuum at room temperature, lyophilised and yields a stronglyhygroscopic, amorphous solid that liquefies on contact with air.

Yield: 222 mg of a colourless oil (99%)

TLC: R_(f) =0.57 (i-propanol/water/acetic acid, 8:1:1; ninhydrine)

¹H-NMR (DMSO-d₆):

δ/ppm=1.20 (t, ³J=7.16 Hz, 3H, CH₂—CH₃), 1.23 (t, ³J=7.13 Hz, 3H,CH₂—CH₃), 1.26 (t, ³J=7.10 Hz, 3H, CH₂—CH₃), 1.51-1.87 (m, 4H, β,γ-CH₂),3.17 (m, 2H, N—CH₂), 3.95 (m, 1H, α-CH), 4.14 (q, ³J=7.12 Hz, 2H,CH₂—CH₃), 4.15 (q, ³J=7.10 Hz, 2H, CH₂—CH₃), 4.17-4.25 (m, 2H, CH₂—CH₃),4.51 (s, 2H, O—CH₂), 7.80 (br s, 1H, NH), 8.55 (br s, 1H, NH), 8.72 (brs, 3H, NH₃ ⁺).

¹³C-NMR (DMSO-d₆):

δ/ppm=13.9, 14.0, 14.1 (3×CH₂—CH₃), 24.2 (γ-CH₂), 27.2 (β-CH₂), 40.8(N—CH₂), 51.5 (α-CH), 60.5, 61.7, 61.9 (3×CH₂—CH₃), 71.1 (O—CH₂), 150.3(C═N), 153.0 (CO—Eoc), 169.1, 169.3 (2×CO).

HRMS (m/z):

calculated for C₁₅H₂₉N₄O₇ [M+H]⁺=377.20308, found: 377.20287.

Exemplary Embodiment 3: O-Glycosidically Conjugated NOHA Derivatives

N^(α)-(t-butyloxycarbonyl)-N^(ω)-ethoxycarbonyl-N^(ω′)-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranos-1-yl)oxy-L-arginine-t-butylester(13)

210 mg of the thiourea 2 (0.5 mmol), 218 mg of1-aminooxy-2,3,4,6-tetra-O-acetyl-β-D-galactopyranose (0.6 mmol) and104.5 μl of DIPEA (0.6 mmol) are dissolved in 5 mL of drydichloromethane. The batch is cooled to 0° C., 115 mg of EDCl (0.6 mmol)are added and stirred at room temperature for 48 hours. The solution isdiluted with approximately 10 mL of dichloromethane and in each casewashed with 5 mL of 1% HCl, water and saturated NaCl solution. Theorganic phase is dried over Na₂SO₄ and concentrated in vacuum. The rawproduct is purified further by means of column chromatography oversilica gel (dichloromethane/methanol, 97:3). The combined fractions ofthe purified product are concentrated in vacuum to form a clear oil.Repeatedly adding and removing dry dichloromethane yields a solid whitefoam that liquefies on contact with air.

Yield: 262 mg of a colourless oil (70%)

TLC: R_(f)=0.35 (dichloromethane/methanol, 97:3; ninhydrine)

¹H-NMR (CDCl₃):

δ/ppm=1.29 (t, ³J=7.1 Hz, 3H, CH₂—CH₃), 1.43, 1.45 (2×s, 9H, C(CH₃)₃),1.52-1.86 (m, 4H, β,γ-CH₂), 1.98, 2.02, 2.05, 2.13 (4×COCH₃), 3.08 (m,2H, N—CH₂), 3.96 (br t, ³J=6.8 Hz, 1H, 5′-CH), 4.11-4.21 (m, 5H, α-CH,CH₂—CH₃, 3′-CH, NH), 4.85 (d, ³J=8.3 Hz, 1H, 1′-CH), 5.06 (m, 2H,6′-CH₂), 5.25 (dd, ³J=10.4, 8.3 Hz, 1H, 2′-CH), 5.39 (dd, ³J=3.5, 1.0Hz, 1H, 4′-CH), 6.47 (br t, 1H, NH), 7.65 (br s, 1H, NH).

MS (ESI):

m/z=772 [M+Na]⁺, 750 [M+H]⁺, 707 [M−C₂H₂O+H]⁺.

Sodium salt of theN^(ω)-ethoxycarbonyl-N^(ω′)-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranos-1-yl)oxy-t-arginine(14)

In a Schlenk tube 120 mg (0.16 mmol) of the completely protected andcarefully dried precursor 13 are dissolved with argon fumigation inapproximately 10 mL of dry diethylether. The solution is stirred forapproximately 30 minutes at −15° C., and then HCl gas is introducedcarefully with simultaneous argon fumigation for approximately 5minutes. The reaction mixture is placed over night in the refrigeratorand concentrated the following day for working up in vacuum. Thewhite-yellow solid is then taken up with approximately 1-2 mL of 0.5 MNaHCO₃ solution and purified by means of flash chromatography over anRP-18 column (flow agent: Aqua bidest./methanol), a step gradient beingused for the elution. The start is made with a concentration of 10%methanol, then increased to 25% and finally to 50% methanol. Theproduct-containing fractions are combined, concentrated at 30° C. invacuum to a residual volume of approximately 50 mL and then lyophilised.

Yield: 74 mg of a fine white powder (75%)

In D₂O as solvent, the substance is present as an isomer mixture in aratio of approximately 6:4 (at 300 K, relative to the triplet of CH₃ ofthe ethoxycarbonyl function). The shifts specified refer to the mainisomer.

¹H-NMR (D₂O, TPS):

δ/ppm=1.28 (t, ³J=7.1 Hz, 3H, CH₂—CH₃), 1.52-1.97 (m, 4H, β,γ-CH₂),2.02, 2.08, 2.12, 2.23 (4×s, 3H, COCH₃), 3.16 (br t, ³J=6.8 Hz, 2H,6′-CH₂), 3.75 (t, ³J=6.3 Hz, 1H, 5′-CH), 4.15-4.30 (m, 5H, CH₂—CH₃,α-CH, N—CH₂), 5.07 (d, ³J=8.0 Hz, 1H, 1′-CH), 5.21-5.33 (m, 2H,2′,3′-CH), 5.47 (m, 1H, 4′-CH).

¹³C-NMR (D₂O, TPS):

δ/ppm=16.2 (CH₂—CH₃), 22.7, 22.8, 22.9, (4×COCH₃), 26.6 (γ-CH₂), 30.6(β-CH₂), 42.7 (N—CH₂), 57.2 (α-CH), 64.6 (6′-CH₂), 65.8 (CH₂—CH₃), 70.6,70.7, 73.4, 74.1 (2′,3′,4′,5′-CH), 104.1 (1′-CH), 155.6 (C═N), 156.8(CO-Eoc), 175.4, 175.8, 176.2, 177.1 (4×COCH₃).

MS (ESIESI):

m/z=615 [M+Na]⁺, 593 [M+H]⁺, 551 [M−C₂H₂O+H]⁺, 331 [C₁₄H₁₉O₉]⁺.

HRMS (m/z):

calculated for C₂₃H₃₃N₄O₁₄Na [M+Na]⁺=615.21202, found: 615.21164

C₂₃H₃₅N₄NaO₁₄ (614.53)

Calculated C 44.95 H 5.74 N 9.12 Found C 45.08 H 6.21 N 8.77

The invention claimed is:
 1. A method of treating a disease accompanyinga nitrogen monoxide deficiency comprising: administering apharmaceutical substance having the general formula (I)

where X₁ is H; X₂ is

R₁ is ethyl; R₂₋₄ is hydrogen, an alkyl, an aryl radical, amonosaccharide or a monosaccharide derivative; Y is hydrogen; n is 2 or3; m is an R or S configured chiral center; and whereN^(ω)-hydroxy-L-arginine and its carboxylic acid ester are exempt, andwherein the disease accompanying a nitrogen monoxide deficiency is acardiovascular disease.
 2. The method according to claim 1, wherein R₂,R₃ and/or R₄ is a monosaccharide or a monosaccharide derivative.
 3. Themethod according to claim 1 wherein the cardiovascular disease isselected from the group consisting of: high blood pressure, heartfailure, stable and unstable angina pectoris, peripheral and cardialvessel diseases, arrhythmias, thromboembolic diseases and ischemiasincluding myocardial infarcation, stroke, transient and ischemicattacks, peripheral circulatory disorders, restenoses after thrombolytictherapies, percutaneous transluminal angioplasties, percutaneoustransluminal coronary angioplasties, or bypass, and atherosclerosis. 4.The method according to claim 1 wherein the pharmaceutical substanceundergoes deprotection and functionalization.
 5. The method of claim 1wherein the pharmaceutical substance is a prodrug of NOHA.